Abstract: According to the World Health Organization, antimicrobial resistance (AMR) among Gram-negative bacteria continues to increase on a global scale. It is estimated that more than 23,000 people in the U.S. alone die each year from infections with multidrug-resistant (MDR) bacteria. New therapeutic agents are critical to stem this trend, but new technologies are required for shortening the time from discovery to production. To support this effort, ATCC has developed a collection of 33 fully characterized Gram-negative isolates representing current MDR disease strains from around the globe. Strains were evaluated using whole genome sequencing (WGS) and a novel annotation program to identify AMR genes and protein targets. Using public databases, ATCC developed and validated an accurate and efficient bioinformatics pipeline for the automated assembly and annotation of microbial genomes. Next-generation sequencing (NGS) data from all 33 individual isolates in combination with the novel bioinformatics pipeline were used to identify AMR genes and predictive targets that could be associated with the observed phenotype. Using our proprietary bioinformatics pipeline, we created a searchable database of AMR determinants containing the WGS information as well as a list of known AMR genes with their corresponding nucleotide sequences.